We will use the modified collagenase perfusion technique that we established in the previous period of the grant in order to isolate and study human hepatocytes in cell culture and in transplantation. Fragments of hepatic tissue will be obtained from scheduled surgical resections and liver needle biopsies. The isolated cells will be studied in primary culture and their rates of protein secretion and the basal and induced levels of microsomal enzymes will be studied. Mixed cultures of human hepatocytes and human fibroblasts will be used for mutagenesis studies. The effects of hepatopoietins from human serum will be also studied on these cells. The effeects of other potential growth modulators in conjunction with the hepatopoietins will be assessed with the objective of establishing conditions that would allow continuous growth of the human hepatocytes. The effects of initiating chemical carcinogens on replicating human hepatocytes will be studied, in view of results with the rat that indicate that this approach is very sensitive for detecting genotoxic effects of chemicals at very low concentrations. Human hepatocytes will be transplanted into nude mouse fat pads for assays of clonogenicity, response to modifiers of xenobiotic metabolism and responses to chemical carcinogens. The clonogenicy of the hepatocytes from the normal and the cirrhotic liver will be compared. This will parallel similar studies in vitro that will monitor the behavior of normal and cirrhotic liver hepatocytes in response to hepatopoietins and different substrata. Human hepatocytes will be also transplanted into the nude mouse spleen. The "hepatized" spleens will be used for studies in carcinogenesis including administration of chemical carcinogens with the purpose of induction of hepatic neoplasia in the transplanted human hepatocytes. In parallel studies human hepatocytes will be injected into the livers of hepatectomized irradiated nude mice with the purpose of producing nude mice with "hybrid" livers that can be used for studies in carcinogenesis and hepatic physiology. A large reservoir of human hepatocytes in cryopreservation is already accumulated and it will be further evaluated with culture and transplantation experiments.